Ten cyclic nucleotide phosphodiesterase gene families (PDE1-10) have been identified. PDE3 isoforms are characterized by their high affinity for cAMP and cGMP; their specific inhibition by certain drugs that increase myocardial contractility, relax airway and vascular smooth muscle, inhibit platelet aggregation and stimulate insulin secretion; and their rapid activation in response to insulin, IGF-1, IL-4, and agents that increase cAMP. A possible pre-translational variant of PDE3A was cloned from a cultured porcine aorta smooth muscle (VSM) cDNA library. The predicted molecular weight of this cardiovascular short PDE3A isoform is similar to that (~110-115 kDa) of a PDE3 purified from bovine aorta. Although we were unable to identify 5 UTR sequence upstream from the putative translation initiation (ATG) codon by 5- RACE, RT-PCR, or primer extension, RNase protection assays indicated the presence of mRNAs corresponding to both short and full-length PDE3A isoforms in myocardium, but only short forms in VSM. To continue our structure/function studies of the N-terminal portion of PDE3, the N- terminal portion was arbitrarily divided into region 1 (aa1-300) and region 2 (aa301-500). Region 1 contains a large hydrophobic domain (of ~200aa) with six predicted transmembrane helical segments. Region 2 contains a smaller hydrophobic domain (of ~50aa). N-terminal truncation mutants, with different amounts of region 1 and region 2 sequence removed, were FLAG-tagged at their C-termini and expressed in COS-7 and Sf9 cells. Subcellular distribution of mutant PDE3 activities in Sf9 cells and immunofluorescent localization in COS-7 cells indicated that the large hydrophobic domain in region 1 contains structural determinants responsible for insertion of PDE3 into, or strong association with, ER membranes. Although region 2, especially the small hydrophobic domain, contains information for targeting of PDE3 to membranous structures, the molecules associate as non-integral membrane proteins that do not anchor efficiently. After removal of regions 1 and 2, PDE3 isoforms were virtually completely localized in the cytosol. We are attempting to learn more of PDE3A and 3B functions by disruption of PDE3A and B genes in mice. Homozygous null PDE3B mice have been generated; functional studies will soon be initiated. The PDE3A gene has been successfully targeted in ES cells and, in collaborative studies, we are attempting to generate PDE3A chimeric mice. In collaborative studies, the human (H) PDE3A gene was cloned; its structural organization is quite similar to that of the HPDE3B gene, which we previously characterized. The intron/exon structure of the catalytic domains of H and M (mouse) PDE3A genes is highly conserved. - PDE3, structure/function, immunofluorescence, membrane association domains, endoplasmic reticulum localization - Human Subjects